Evaluation of the effect of colloidal systems on biodistribution of selected prostate cancer radiopharmaceuticals

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Evaluation of the effect of colloidal systems on

biodistribution of selected prostate cancer

radiopharmaceuticals

V Mandiwana

orcid.org / 0000-0002-2647-9780

Thesis submitted in fulfilment of the requirements for the

degree

Doctor of Philosophy in Pharmaceutics

at the

North-West University

Promoter: Prof JR Zeevaart

Co-Promoter: Prof RK Hayeshi

Co-Promoter: Mr ML Kalombo

Graduation: October 2019

Student number: 24045756

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PREFACE

This thesis is submitted in fulfilment of the requirements of a Doctor of Philosophy in Pharmaceutics using the article format in accordance with the General Academic Rules (A.7.5.7.4) of the North-West University. Each experimental chapter was written in accordance with specific guidelines as stipulated by the journals intended for publication.

I Vusani Mandiwana, the student did the following work: • Planned and designed the experiments

• Carried out and participated in all the experiments with the exception of analysis done at independent laboratories

• Interpreted the results and discussed them with various co-authors and/or supervisors • Drafted the manuscripts

Manuscript 1 has been accepted for publication in the Journal of Labelled Compounds and Radiopharmaceuticals, manuscript 2 and manuscript 3 will be submitted to the Journal of Nanomaterials and Journal of Nanoparticle Research respectively.

All the co-authors have given permission that the manuscripts may be submitted for degree purposes as stipulated in the Manual for Post Graduate Students of the North-West University.

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ACKNOWLEDGEMENTS

I would love to express my sincerest gratitude to the following people and institutions, all of who played a significant role during the course of my PhD study.

Prof Jan Rijn Zeevaart, my supervisor, thank you so much for allowing me the opportunity to complete a study such as this under your supervision. Your knowledge, expert advice and suggestions made this research so much easier. Thank you for all the assistance, support and encouragement throughout my study and continuously being patient with me.

Mr Lonji Kalombo, my co-supervisor, thank you for being my father in science. Your motivation, words of wisdom and supervision/mentorship will forever be appreciated. Thank you for believing in me, for boosting my confidence when I doubted my abilities, for giving me a platform and continuously encouraging me to do better.

Prof Rose Hayeshi, your drive and efficiency is just amazing, it inspired me to work harder and it often made me feel guilty when I was slacking. Thank you most especially for your patience. Dr Thomas Ebenhan, thank you for your valuable input in my experimental work and all your plan B’s. I was never short of supervisors thanks to you coming on board to assist and advice whenever and wherever possible.

Dr Yolandy Lemmer, thank you for all your assistance, the sisterly advice, your emotional support and your scientific contribution to my study, you make science so enjoyable. Thank you for your loving and motivating energy, it always encouraged me.

Prof Anne Grobler, North West University, Preclinical Drug Development Platform (NWU/PCDDP), thank you for your helpful suggestions and input in my research. Your passion for research is inspiring.

Prof Mike Sathekge, thank you for welcoming me into your department with open arms and allowing me access to work and learn from your staff and lab facilities. Thank you for the opportunity to learn and grow as a scientist.

Dr Hester Oosthuizen, thank you for your assistance, advice and all the input you made in putting my thesis together. You made the writing up process a breeze and I appreciate all your assistance.

All my colleagues, at the Council for scientific and industrial research (CSIR), Nuclear energy corporation of South Africa (Necsa), NWU/PCDDP and the Steve Biko Academic Hospital

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Department of Nuclear Medicine, thank you for all your input and assistance in my research, for challenging me and all the fun times. Your contributions made my study so much easier. Your efforts, time and various contribution made my study possible.

To my team at CSIR, I would like to extend my gratitude to Phuti Chelopo, Heena Ranchod, Patric Nkuna, Nthwaleng Mogamme, Bathabile Ramalapa, Philip Labuschagne, Andri Swanepoel.

Thank you to all my friends and colleagues at NWU/PCDDP namely Antoinette Fick, Cor Bester, Kobus Venter, Jacob Mabena, Nico Minnaar, Hyltton Bunting, Liezl-Marie Scholtz, Zaan Welgemoed, Matthew Glyn, Adelle As, Ambrose Okem, Palesa Koatale, Lerato Thindisa, Magda Lombard.

Team Necsa and Steve Biko Academic Hospital, I would like to thank the following people; Brenda Mokaleng for teaching me so much on radiochemistry and being patient with me, Janke Kleinhans, Amanda Mdlophane, Cathryn Driver, Janie Duvenhage, Mariana Miles, Collins Maledimo, Johncy Mahapane, Thato Sello, Delene van Wyk, Cindy Els, Danka Erasmus (Axim), Nadia Capelo (Axim) and Adolf Nordin (Axim).

My family has been my saving grace, thank you to my parents Emma and Jeffrey Mandiwana for your constant motivation, love and support in everything I pursued. Your faith in me drives me to do better and be better in everything I do and it helped me to reach higher without giving up, “ndo livhuwa”. My sisters Onica, Sedzani and Mpho, thank you for making me feel like a super-woman who can do anything no matter how tough it may seem. Your support and encouragement is always highly appreciated. I love you girls. To my sons, Andani and Londani, I did this for you guys. You’ve always been my inspiration, the reason, my purpose and my driving force. I love you both so much.

Nuclear technologies in medicine and the biosciences initiative (NTeMBI), thank you for hosting me and for the financial support. I would like to extend my sincerest gratitude for allowing me this opportunity to be a part of a great team of institutions and to complete my study. A special thank you to the Carl and Emily Fuchs Foundation for assisting in funding my PhD, thank you, thank you, thank you.

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ABSTRACT

It has been reported that microemulsion (ME) delivery systems provide an opportunity to enhance the bioavailability and efficacy of a therapeutic agent whilst minimising side effects. The prostate-specific membrane antigen (PSMA) targeting agents PSMA-11 and PSMA-617, which accumulate in prostate tumours, allows for [68Ga]Ga3+-radiolabelling and PET imaging of PSMA-expression

in vivo. Radiolabelled [68Ga]Ga-PSMA-617 can be encapsulated in a ME delivery system which is hypothesized to enhance its pharmacokinetic properties. This study investigated the synthesis of [68Ga]Ga-PSMA-617 and [68Ga]Ga-PSMA-617 contained within a ME, the toxicity profile, and microPET/CT imaging and biodistribution in PC3 tumour xenograft male BALB/c mice. [68Ga]Ga-PSMA-617 was synthesized in a combined solid phase and solution chemistry strategy. The formulation of [68Ga]Ga-PSMA-617 into a ME was then evaluated for in vitro and in vivo toxicity and biodistribution. The cytotoxicity of [68Ga]Ga-PSMA-617-ME was tested in HEK293 and PC3 cells. [68Ga]Ga-PSMA-617-ME indicated negligible cellular toxicity at different concentrations. HEK293 cells showed a statistically higher tolerance towards the [68 Ga]Ga-PSMA-617-ME compared to PC3 cells. [68Ga]Ga-PSMA-617 and [68Ga]Ga-PSMA-617-ME was administered intravenously in BALB/c mice with or without PC3-tumours followed by microPET/CT imaging and ex vivo biodistribution determination. The ex vivo biodistribution in PC3-tumour bearing BALB/c mice showed the highest amounts of [68Ga]Ga-PSMA-617 radioactivity accumulation in the kidneys and the lowest uptake was seen in the brain. Both the [68Ga]Ga-PSMA-617 and [68Ga]Ga-PSMA-617-ME followed an expected clearance profile for small-sized polar radiopharmaceuticals, with predominant renal clearance. The ME did alter the biodistribution pattern of [68Ga]Ga-PSMA-617 but maintained distribution to the kidneys, albeit at statistically significant higher levels. Similarly, encapsulation in the ME may have resulted in delayed uptake into tumours as can be seen from the higher blood pool values.

The incorporation of [68Ga]Ga-PSMA-617 into ME was successfully demonstrated and resulted in a stable non-toxic formulation as evaluated by in vitro and in vivo means. Both the [68 Ga]Ga-PSMA-617 and [68Ga]Ga-PSMA-617-ME showed enterohepatic metabolism of [68 Ga]Ga-PSMA-617.

Keywords

Biodistribution, Ex vivo, 68Ga-PSMA-617, In vitro, In vivo, Microemulsion, MicroPET/CT imaging, Prostate cancer, Toxicity

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Chapter 1: Problem Statement

1.1. Introduction

Prostate cancer is the second most frequent cancer and the sixth leading cause of cancer deaths in men worldwide (Afshar-Oromieh et al., 2014; Akhtar et al., 2012; Sathekge et al., 2018). It is a challenge to select appropriate treatment options for disseminated prostate cancer due to the lack of sensitive imaging agents for diagnosis and therapy monitoring (Eder et al., 2013). Prostate-specific membrane antigen (PSMA) is expressed in nearly all prostate cancers with increased expression in poorly differentiated, metastatic and hormone-refractory carcinomas (Eder et al., 2013). PSMA is a cell surface membrane-type zinc protease, also called glutamate carboxypeptidase II (GCPII) (Eder et al., 2014) and is primarily restricted to the prostate. It is expressed in all stages of the disease, on the tumour cell surface and not shed into the circulation (Eder et al., 2013). PSMA exhibits very high expression in prostate cancer cells compared to other PSMA expressing tissues such as the small intestine, kidneys or salivary glands (Afshar-Oromieh et al., 2013; Baur et al., 2014). PSMA is a transmembrane protein with a large extracellular domain (Afshar-Oromieh et al., 2015). Its enzyme activity permits the development of specific inhibitors and their internalization after binding to a ligand (Afshar-Oromieh et al., 2013). This characteristic thus makes it an ideal and a promising target for prostate cancer-specific imaging and therapy (Eder et al., 2013). Linking of PSMA ligands to small molecules labelled with a positron emitting radionuclide is a potential approach for the diagnosis of prostate cancer using positron emission tomography (PET).

Methods have been developed to label PSMA ligands with gallium-68([68Ga]Ga3+) or 177Lu, enabling their use for PET (Srirajaskanthan et al., 2010) or single photon emission computed tomography (SPECT) imaging and therapy respectively (Afshar-Oromieh et al., 2014). Experience with positron emission tomography/computed tomography (PET/CT) using Glu-NH-CO-NH-Lys-(Ahx)-[68Ga-N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl] ethylenediamine-N,N'-diacetic acid (HBED-CC)] ([68_Ga]Ga3+_{-PSMA-11) as a [}68_Ga]Ga3+_{-labelled PSMA}

ligand, suggests that this tracer can detect prostate cancer relapses and metastases with high contrast by binding to the extracellular domain of PSMA, followed by internalization (Afshar-Oromieh et al., 2014). SPECT is usually performed using a rotating gamma camera system (Perkins and Frier, 2004), whereas PET is performed on a dedicated PET scanner comprising a circular array of detectors which looks more like a computed tomography scanner

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(Afshar-Oromieh et al., 2014). A PET scan looks at the biological activity of a tumour, whereas a CT scan gives anatomical information such as the exact location, size and shape of a tumour in relationship to other structures. PET scanners have a higher spatial resolution than SPECT systems (Afshar-Oromieh et al., 2013).

There is a need to develop high-resolution PET imaging technologies using the extracellular domain of PSMA. Methods have been developed to label PSMA ligands with radionuclides such as [68_Ga]Ga3+_{, technetium-99m (}99m_{Tc) and iodine-123/131 (}123/131_{I), to enable their use}

in scintigraphic imaging and radioligand therapy (Afshar-Oromieh et al., 2013). Of these, [68Ga]Ga3+ is the most widely used radionuclide. It is a positron emitter with a half-life of 68 min. Germanium-68/Gallium-68 (68Ge/68Ga) generators are commercially available, which enable convenient elution of cationic [68Ga]Ga3+ with dilute acid (0.1 M HCl). An effective purification of the 68Ge/68Ga eluate (Asti et al., 2008), a crucial step for clinical use (Zhernosekov et al., 2007), was applied to the production of 68 Ga-1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetraacetic acid–D-Phe1-Tyr3-octreotide (68Ga-DOTATOC), the most widely used [68Ga]Ga3+-based PET radiopharmaceutical (Prata, 2012; Singh et al., 2011). This purification technique has also been used to optimise [68Ga]Ga3+-labelling methods for 1,4,7,10-tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid (DOTA)-derived peptides. Decristoforo et al. (2007) described a fully automated synthesis for [68Ga]Ga3+-labelled peptides with high, reproducible yields. 68 Ga-1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetraacetic acid–D-Phe1-Tyr3-octreotate (68Ga-DOTATATE) is used for neuroendocrine tumour diagnosis using PET/CT. 68Ga-DOTATATE PET/CT has a much higher sensitivity compared to indium-111 octreotide imaging. 68_{Ga-DOTATATE is a conjugate of an amide of}

the acid DOTA, which acts as a chelator for a radionuclide, and (Tyr3)-octreotate, a derivative of octreotide. The latter binds to somatostatin receptors, which are found on the cell surfaces of a number of neuroendocrine tumours, and thus directs the radioactivity into the tumour.

PSMA catalyses the hydrolysis of aspartyl-L-glutamate (NAAG) into N-acetyl-L-aspartate (NAA) and L-glutamate (Baur et al., 2014). Based on the chemical structure of NAAG, several glutamate-urea-glutamate-based peptides have been developed (Baur et al., 2014). These molecules have a high affinity and specific binding to PSMA and differ mainly in the selection of the chelator for the complexation of the desired radionuclide (Baur et al., 2014). Due to their good affinity for PSMA, it is therefore desirable to develop radionuclide

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In recent years 68Ga-DOTA-PSMA (Figure 1) conjugates have been used as a diagnostic tool to detect tumour lesions. However, the application of lutetium-177 (177Lu) and yttrium-90 (90Y) is favoured for radiometal therapy (Kam et al., 2012). This means that [68Ga]Ga3+-PSMA can be used to diagnose prostate cancer followed by further diagnosis and/or treatment therapy using 177Lu-DOTA-PSMA (177Lu-PSMA) applications. The use of 177Lu (Eβmax: 0.5 MeV, t1/2:

6.7 d) is more suitable for the treatment of smaller lesions and metastases, accompanied by a minimization of kidney dose in comparison to the application of 90_{Y labelled peptides (Baur et} al., 2014). Yttrium-90 (Eβmax: 2.3 MeV, t1/2: 64 h) is more appropriate for the treatment of

larger tumour lesions and metastases (Baur et al., 2014). 177Lu is also a useful diagnostic tool for the scintigraphy of tumour uptake due to beta and gamma emission (Baur et al. 2014). For both targeting and treatment applications, DOTA (Kam et al., 2012) is the most commonly used chelator for the complexation of radiometals (i.e. 68Ga, 177Lu, and 90Y) to small molecules and peptides (Baur et al., 2014).

Figure 1.1: Representation of a gallium-68 labelled DOTA conjugate of a PSMA targeting

molecule

Theranostics is a term which refers to the inseparability of diagnosis and therapy and takes into account the personalised management of the disease for a specific patient (Baum and Kulkarni, 2012; Werner et al., 2014). Theranostic radionuclides emit radiation energy suitable for both diagnostic and treatment purposes due to the physical behaviour of the decay of the parent nuclide during the emission of gamma, beta and alpha radiation. Molecular phenotypes of

68

Ga

PSMA

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tumours can be determined by molecular imaging, using techniques such as PET, SPECT, magnetic resonance imaging (MRI) or optical methods so that the treatment is specifically targeting the tumour. To meet the demands of theranostics, the target, ligand, labelling chemistry, the most appropriate radionuclide, biodistribution modifier and the patient for the personalised treatment need to be defined (Baum and Kulkarni, 2012). Theranostics of neuroendocrine tumours using [68Ga]Ga3+-labelled tracers for diagnostics with PET/CT and using 177_{Lu or other radionuclides for radionuclide therapy by applying the same peptide is}

proving to be useful (Baum and Kulkarni, 2012). Although treatment with 177_{Lu has shown}

good response, side effects such as renal impairment with a decline in creatinine clearance has been reported (Werner et al., 2014). Amino acid solutions are recommended directly prior and during peptide receptor radionuclide therapy (PRRT) to reduce the renal absorbed dose and damage to the renal parenchyma (Werner et al., 2014).

The use of delivery systems (such as microemulsions) can improve the efficacy of a drug, allowing the total dose to be reduced and thus minimising side effects and toxicity of the encapsulated compound (Muzaffar et al., 2013).

Microemulsions (MEs) are a macroscopic system of water, oil and amphiphile which is optically isotropic (Muzaffar et al., 2013). MEs are clear or translucent and form spontaneously with an average droplet diameter of 10 to 140 nm (Muzaffar et al., 2013). They are thermodynamically stable and nanostructured (Moghimipour et al., 2013). This distinguishes them from ordinary milk-like emulsions which are thermodynamically unstable but kinetically stable. The advantages of MEs include enhanced bioavailability of the encapsulated compound, improved solubility of a poorly soluble drug, protection of unstable drugs against environmental conditions and a long shelf life. MEs can be classified as water-in-oil (w/o), oil-in-water (o/w) or bicontinuous phase MEs (Moghimipour et al., 2013).

This study aims to formulate and characterize a ME as an intravenous delivery system of [68_Ga]Ga3+_{-labelled PSMA conjugated with the chelator DOTA (this will be referred to as}

[68Ga]Ga-PSMA-617) for the diagnosis of prostate cancer. The ME will serve to encapsulate the radiopharmaceutical ([68Ga]Ga-PSMA-617) to assist in a rapid release of the radiopharmaceutical into the target organ (prostate) while altering the clearance mechanism of peptides away from rapid kidney clearance towards excretion via the liver and bile.

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1.2 Research Question

Mortality from metastasising prostate cancer is still high despite the use of novel therapeutic approaches (Baur et al., 2014). There is therefore an urgency to diagnose prostate cancer early to prevent tumour dissemination. Furthermore, effective strategies for disseminated prostate cancer are needed urgently. Selective targeting of prostate cancer or its metastases is a significant task in molecular imaging with PET and for targeted internal radiation therapy.

Scintigraphy techniques (gamma camera) usually show lower spatial resolution when compared to PET. Furthermore, imaging with radiolabelled compounds, for example antibodies, is relatively complex: after injection, multiple acquisitions over several days are needed to obtain the best images with high contrast between background and tumour lesions (Afshar-Oromieh et al., 2013).

68_{Ga-DOTATATE on the other hand is rapidly excreted from non-target sites, offers a good}

target to background ratio and has documented efficacy for imaging neuroendocrine tumours (Shastry et al., 2010). However, evaluation of a lesion with low or atypical uptake of 68 Ga-DOTATATE is a challenge, especially if the lesion is located in an organ showing physiological uptake, such as the kidneys, adrenals, spleen or bowels.

Significant accumulation of [68Ga]Ga3+ radioactivity levels in tumours, kidneys and the bladder after in vivo administration of 68Ga-DOTATATE in mice has been reported (Müller, 2013). The kidneys tend to have a very high uptake of radioactive [68_Ga]Ga3+_{, with a high and fatal}

risk of dysfunction. Whole body SPECT/computed tomography (SPECT/CT) image scans with 177_{Lu-DOTATATE have also reportedly shown high physiological uptake in the spleen,}

kidneys, bladder and increased abnormal uptake in the liver (Singh et al., 2011). Therefore, the design and development of drug delivery systems with the intention of enhancing drug availability while minimising accumulation in kidneys and other non-target tissues and subsequent side effects are an ongoing process in pharmaceutical research. Despite abnormal uptake and risk of nephrotoxicity, the prediction of therapeutic response would be more accurate based on 177Lu-DOTATATE diagnostic or therapy scans as compared to other 177 Lu-labelled somatostatin analogues (Poeppel et al., 2011; Singh et al., 2011) and therefore 177 Lu-labelled PSMA.

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The use of colloidal particles as delivery systems can improve the efficacy of the drug, allowing the total dose to be reduced and therefore minimizing side effects (Bhattacharya et al., 2016).

This study will attempt to answer the following questions:

The advantages of MEs over other emulsions include short preparation time, thermodynamic stability, increased drug loading, increased bioavailability and enhanced penetration through the biological membranes. These are versatile delivery systems which can be used to deliver therapeutic compounds via several routes. Considering these advantages, can

i.) this delivery system lead to the rapid absorption of an encapsulated therapeutic compound,

ii.) reduce the toxic side effects of the drug while being maintained within the efficacious concentrations for a prolonged period in the blood stream?

This approach to encapsulate [68Ga]Ga-PSMA-617 prostate cancer targeting compounds into a ME delivery system could lead to optimised delivery systems with decreased toxicity and reduced accumulation of the compound in the kidneys. The ME delivery system will aid to make the diagnostic and therapeutic compound which has been encapsulated, safe in vivo due to reduced toxicity and side effects to the kidneys and other non-target organs. These delivery systems will aid to distribute the entrapped radioactive [68Ga]Ga-PSMA-617 to the prostate cancer while reducing toxic uptake or accumulation in the kidneys. In this study, the following hypothesis should be tested:

a) The use of MEs as delivery systems can improve the efficacy of a drug or encapsulated therapeutic compound, allowing the total dose to be reduced and thus minimising side effects.

b) The development of this characteristic nano-sized delivery system will minimize toxicity risks by reducing the duration of exposure to the radioactive, entrapped compound (Longmire et al., 2008). Additionally, nano-sized particles with characteristic size, shape, charge, polarity and lipophilicity will show tumour uptake to a certain extent via the enhanced permeability and retention (EPR) effect (Heneweer et al., 2011).

Therefore, the expected outcomes of this delivery system are:

 Enhanced bioavailability and increased absorption rates of [68_{Ga]Ga-PSMA-617, without}

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 Improved intracellular delivery of the encapsulated [68_{Ga]Ga-PSMA-617 into the prostate}

cancer.

1.3 Aim and research objectives

The aim of this study was to initially encapsulate a [68Ga]Ga3+-labelled PSMA ligand in a ME and to investigate the uptake of this PSMA formulation in healthy and mice with human tumour xenografts to evaluate its applicability for prostate cancer or tumour imaging vs the non-encapsulated [68Ga]Ga-PSMA-617. This encapsulated radiopharmaceutical complex will serve as a diagnostic tool. Therefore, the specific objectives of this study are as follows:

i. To produce a radiolabelled ME

 Encapsulate [68Ga]Ga-PSMA-617 in a ME delivery system for diagnosis of prostate cancer.

ii. To evaluate the impact of the ME on the biodistribution of [68_{Ga]Ga-PSMA-617 as}

measured by microPET after IV administration.

 Assess the biodistribution of [68Ga]Ga-PSMA-617 encapsulated in a ME delivery system ( henceforth [68Ga]Ga-PSMA-617-ME) in healthy male BALB/c mice.  Compare the biodistribution of [68Ga]Ga-PSMA-617-ME vs non-encapsulated

[68Ga]Ga-PSMA-617 in prostate cancer PC3 tumour bearing male BALB/c mice. iii. Evaluate the in vitro cellular toxicity of [68Ga]Ga-PSMA-617-ME and [68

Ga]Ga-PSMA-617 to PSMA-positive PC3 and PSMA-negative HEK293 cell lines.

iv. Evaluate the in vivo toxicity of a ME delivery system and of a ME containing [68

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References

Afshar-Oromieh A, Malcher A, Eder M, Eisenhut M, Linhart HG, Hadaschik BA, Holland-Letz T, Giesel FL, Kratochwil BA, Haufe S, Haberkorn U and Zechmann CM. 2013. PET imaging with a [68Ga]gallium-labelled PSMA ligand for the diagnosis of prostate cancer: Biodistribution in humans and first evaluation of tumour lesions. European

Journal of Nuclear Medicine and Molecular Imaging 40: 486–495

Afshar-Oromieh A, Zechmann CM, Malcher A, Eder M, Eisenhut M, Linhart HG, Holland-Letz T, Hadaschick BA, Giesel FL, Debus J and Haberkorn U. 2014. Comparison of PET imaging with a 68Ga-labelled PSMA ligand and 18F-Choline-based PET/CT for the diagnosis of recurrent prostate cancer. European Journal of Nuclear Medicine and

Molecular Imaging 41: 11–20

Afshar-Oromieh A, Hetzheim H, Kratochwil C, Benesova M, Eder M, Neel OC et al. 2015. The novel theranostic PSMA-ligand PSMA-617 in the diagnosis of prostate cancer by PET/CT: biodistribution in humans, radiation dosimetry and first evaluation of tumor lesions. Journal of Nuclear Medicine 56(11)

Akhtar NH, Pail O, Saran A, Tyrell L and Tagawa ST. 2012. Prostate-specific membrane antigen-based therapeutics. Advances in Urology 2012: Article ID 973820

Asti M, De Pietri G, Fraternali A, Grassi E, Sghedoni R, Fioroni F, Roesch F, Versari A and Salvo D. 2008. Validation of 68Ge/68Ga generator processing by chemical purification for routine clinical application of 68Ga-DOTATOC. Nuclear Medicine and Biology 35: 721–724

Baum RP and Kulkarni HR. 2012. Theranostics: From molecular imaging using Ga-68 labeled tracers and PET/CT to personalized radionuclide therapy - The Bad Berka Experience.

Theranostics 2 (5): 437–447

Baur B, Solbach C, Andreolli E, Winter G, Machulla HJ and Reske SN. 2014. Synthesis, radiolabelling and in vitro characterization of the gallium-68-, yttrium-90- and

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lutetium-Bhattacharya R, Mukhopadhyay S and Kothiyal P. 2016. Review on microemulsion- As a potential novel drug delivery system. World Journal of Pharmacy and Pharmaceutical

Sciences 5(6): 700-729

Eder M , Neels O, Müller M, Bauder-Wüst U, Remde Y, Schäfer M, Hennrich U, Eisenhut M, Afshar-Oromieh A, Haberkorn U and Kopka K. 2014. Novel preclinical and radiopharmaceutical aspects of [68_{Ga]Ga-PSMA-HBED-CC: A new PET tracer for}

imaging of prostate cancer. Pharmaceuticals 7: 779–796

Eder M, Eisenhut M, Babich J and Haberkorn U. 2013. PSMA as a target for radiolabelled small molecules. European Journal of Nuclear Medicine and Molecular Imaging 40: 819–823

Kam BLR, Teunissen JJM, Krenning EP, De Herder WW, Khan S, Van Vliet EI and Kwekkeboom DJ. 2012. Lutetium-labelled peptides for therapy of neuroendocrine tumours. European Journal of Nuclear Medicine and Molecular Imaging 39(1): S103-S112

Moghimipour E, Salimi A and Eftekhari S. 2013. Design and characterization of microemulsion systems for naproxen. Advanced Pharmaceutical Bulletin 3(1): 63–71

Müller C. 2013. Folate-based radiotracers for PET imaging-update and perspectives. Molecules 18: 5005–5031

Muzaffar F. Singh UK and Chauhan L. 2013. Review on microemulsion as futuristic drug delivery. International Journal of Pharmacy and Pharmaceutical Sciences 5(3): 39–53

Poeppel TD, Binse I, Petersenn S, Lahner H, Schott M, Antoch G, Brandau W, Bockisch A and Boy C. 2011. 68Ga-DOTATOC versus 68Ga-DOTATATE PET/CT in functional imaging of neuroendocrine tumors. Journal of Nuclear Medicine 52 (12): 1864–1870

Prata IM. 2012. Gallium-68: A new trend in PET radiopharmacy. Current

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Sathekge M, Lengana T, Maes A, Vorster M, Zeevaart JR, Lawal I et al. 2018. 68 Ga-PSMA-11 PET / CT in primary staging of prostate carcinoma : preliminary results on differences between black and white South-Africans. European Journal of Nuclear Medicine and

Molecular Imaging 45: 226–234

Shastry M, Kayani I, Wild D, Caplin ME, Visvikis D, Gacinovic S, Reubi JC and Bomanji JB. 2010. Distribution pattern of 68_{Ga-DOTATATE in disease-free patients. Nuclear} Medicine Communications 31: 1025–1032

Srirajaskanthan R, Kayani I, Quigley AM, Soh J, Caplin ME and Bomanji JB. 2010. The Role of 68Ga-DOTATATE PET in patients with neuroendocrine tumors and negative or equivocal findings on 111In-DTPA-octreotide scintigraphy. Journal of Nuclear

Medicine 51: 875–882

Werner RA, Bluemel C, Allen-Auerbach MS, Higuchi T and Herrmann K. 2014. 68Gallium- and 90yttrium-/177lutetium: ‘Theranostic twins’ for diagnosis and treatment of NETs.

Annals of Nuclear Medicine 29: 1–7

Zhernosekov KP, Filosofov DV, Baum RP, Aschoff P, Bihl H, Razbash AA, Jahn M, Jennewein M and Rösch F. 2007. Processing of generator-produced 68Ga for medical application. Journal of Nuclear Medicine 48: 1741–1748

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Chapter 2: Literature Review

2.1 Prostate cancer

The prostate gland (Figure 2.1) is a small, round pea or walnut-sized (in adult life) gland that lies in the pelvic cavity in front of the rectum and slightly below the bladder (Kgatle et al., 2016). The prostate gland is a gland found only in men and serves to produce seminal fluid that assists in transporting sperm during ejaculation (Kgatle et al., 2016).

Figure 2.1: Diagram showing the position of the prostate gland in the male reproductive

system

Prostate cancer is a type of cancer which begins when cells in the prostate gland start to grow uncontrollably (Kgatle et al., 2016; Sathekge et al., 2018(a)). It is caused by changes in the deoxyribonucleic acid (DNA) of normal prostate cells (Kgatle et al., 2016). In normal cells, the fate of damaged DNA would normally result in cell death or its repair (Kgatle et al., 2016). However, in cancer cells, the cells do not die or get repaired as they should be. These cells then continue replicating into cells that the body does not need (Kgatle et al., 2016) and no longer function as healthy cells. Cancerous prostate cells exhibit the following; abnormal structure, uncontrolled growth and the ability to move to other parts of the body (invasive) (Kgatle et al., 2016). Rectum Bladder Prostate gland Urethra Epididymis Testis

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Prostate cancer is one of the most prevalent cancers in males worldwide and also one of the leading causes of morbidity and mortality globally (Emmett et al., 2017; Zhu et al., 2016) and also in South Africa (Sathekge et al., 2018(a)). Epidemiological studies demonstrate vital differences in incidence and clinical behaviour of prostate cancer among patients in South Africa (Vorster et al., 2015; Sathekge et al., 2018(a)). Race is a risk factor and the incidence and mortality rate in black men is almost twice that of white men (Sathekge et al., 2018) and five times higher than that of Asian men (Hsing et al., 2015; Tindall et al., 2014;Vorster et al., 2015). Therefore black men are at higher risk of developing prostate cancer (Table 2.1) and often develop an aggressive type of prostate cancer (CANSA, 2016; Kgatle et al., 2016; Sathekge et al., 2018(a); Vorster et al., 2015).

Table 2.1: The incidence of prostate cancer in South Africa according to the National Cancer

Registry (CANSA, 2016).

Group - Males 2010

No of Cases Lifetime Risk Percentage of All Cancers All males 4 652 1:27 17,15% Asian males 144 1:32 19,65% Black males 2 050 1:39 19,34% Coloured males 518 1:21 16,22% White males 1 940 1:15 15,39%

At a molecular level, the development of prostate cancer may be a result of a complex interaction between important genetic and cellular factors ( Afaq et al., 2018; Kgatle et al., 2016; Vorster et al., 2015).

2.2 Risk factors of prostate cancer

The risk factors of prostate cancer would be anything that affects the chances of a male getting the disease. Risk factors do not necessitate acquiring the disease. Some men may exhibit more than one risk factor and never get the disease whereas other men may have few or no known risk factors and still get cancer. Some of the known risk factors of prostate cancer are discussed in the following paragraphs.

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2.2.1 Age

The chances of developing the disease increases as men become older. The risk rises after the age of 50 (Sathekge et al., 2018(a)) with most cases of prostate cancer diagnosed in men over the age of 65 years (Kgatle et al., 2016).

2.2.2 Race

Prostate cancer is reported to be more common in black or African men as compared to Asian and Caucasian males (Sathekge et al., 2018(a)). It is likely that hormonal, dietary and molecular factors may contribute significantly to the racial disparity of prostate cancer in black African men (Sathekge et al., 2018(a)). It is also suggested that the racial differences in the cancer profile may be the cause for faster prostate cancer growth and earlier transformation from indolent to aggressive prostate cancer in black men as compared to Caucasians (Sathekge

et al., 2018(a)). When looking at nationality, prostate cancer is less common in Asia, Central

America and South America (Kgatle et al., 2016). It is however, most common in North America, North-western Europe, Australia and the Caribbean Islands (American Cancer Society).

2.2.3 Family history

The risk of developing prostate cancer is most probably higher for men with several affected relatives, particularly a father or brother who was diagnosed at a young age (Kgatle et al., 2016). The observation that prostate cancer seems to run in some families suggests that in some cases it may be inherited or genetically acquired (Kgatle et al., 2016; Sathekge et al., 2018(a)).

2.2.4 Nutrition and lifestyle

There are a few lifestyle choices that could either increase or decrease a person’s chances of developing prostate cancer, such as smoking, high alcohol intake, exercise and a good diet (Kgatle et al., 2016; Vorster et al., 2015). With regards to smoking, most studies have not yet found the link between smoking and the risk of developing prostate cancer. Some studies have found that obese men have a higher risk of developing an aggressive form of prostate cancer

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and dying from it, but have a lower risk of developing a less dangerous form of the cancer (Kgatle et al., 2016). Men with a high consumption of red meat and /or high-fat dairy products have an increased chance of developing prostate cancer (Kgatle et al., 2016). Processed food, whole-milk dairy products and fatty meat contain a lot of saturated fat which increases the production of testosterone, which may subsequently assist the growth of prostate cancer cells (Kgatle et al., 2016).

2.3 Detection of prostate cancer

2.3.1 Signs and symptoms of prostate cancer

Prostate cancer does not always show any symptoms in its early stages, thus necessitating annual screening for early diagnosis. Advanced prostate cancer can cause some of the following symptoms (Kgatle et al., 2016):

 Blood in the urine (haematuria)

 Impotence or difficulty getting an erection

 Difficulty urinating, increased urinating frequency especially at night and a slow or weak urinary stream

 Loss of bladder or bowel control

 Numbness or weakness in the legs and/or feet

 Pain in the chest (ribs), back (spine) and hips especially if the cancer has spread to bones

These symptoms may be caused by prostate cancer but can more often than not also be caused by other conditions that are not cancer, most commonly benign prostatic hyperplasia (BPH) (Kgatle et al., 2016). Some of the symptoms are experienced as a result of the prostate gland becoming enlarged and pressing on and blocking the urethra and bladder (UCSF Medical Center; Kgatle et al., 2016). This enlargement of the prostate happens as men age (Kgatle et

al., 2016).

2.3.2 Prostate-specific antigen test

The prostate-specific antigen test is usually used to detect prostate cancer early in men who may not have symptoms. It is however also one of the first tests which are done in patients who have symptoms that may be caused by prostate cancer (Sathekge et al., 2018(a)).

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Prostate-Specific Antigen (PSA) is a protein which is produced by prostate cells and is found in the blood (Sathekge et al., 2018(a)). It represents both the volume of the normal and cancerous prostate cells (Kgatle et al., 2016; Sathekge et al., 2018(a)). The higher the level of PSA, the more likely it is that the person may have prostate cancer (Sathekge et al., 2018(a); Vorster et

al., 2015). PSA test results are reported as nanograms per millilitre (ng/ml) (Kgatle et al.,

2016). In previous years, PSA results of 4.0 ng/ml or below were considered normal, and above 4.0 ng/ml were considered high (UCSF Medical Center). Recent research has shown that prostate cancer can be detected at all PSA levels, however, the chances of detecting prostate cancer increases as PSA increases (Kgatle et al., 2016; Vorster et al., 2015; Sathekge

et al., 2018(a)). PSA levels may also increase with age and prostate size (Sathekge et al.,

2018(a)). There are a few conditions or activities which can cause the production of high PSA, such as (Kgatle et al., 2016):

 An acute urinary tract infection  Benign prostatic hyperplasia

 Ejaculation up to three days prior to testing

 A recent prostate biopsy (the patient should wait at least six weeks after a prostate biopsy before testing for PSA levels)

 Prostatitis (an inflammation of the prostate that was treated successfully with antibiotics)  Bicycle riding (very rare cases)

Similarly, low or normal PSA levels do not imply that prostate cancer is not present (Sathekge

et al., 2018(a)). Results from other tests such as a digital rectal examination (DRE), a colour

Doppler transrectal ultrasound (TRUS), the percentage free-PSA and the PSA velocity should also be considered to make an assessment (Ebenhan et al., 2015). Prostate cancer in some men produces very little PSA. There are certain herbal preparations and medication which may lower PSA levels and thus possibly mask the presence of prostate cancer, especially in its early stages of development (Kgatle et al., 2016; UCSF Medical Center). These may include Estrogens, Finasteride (Proscar or Prospecia), Dutasteride (Avodart or Avocar), Saw palmetto (herb used to treat benign prostatic hyperplasia) and herbal mixtures such as Prostasol (UCSF Medical Center).

PSA tests are most commonly used for the early detection of prostate cancer but it also plays an important role in other situations (Vorster et al., 2015). PSA tests are used to monitor the

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effectiveness of prostate cancer treatment (during and after treatment) and should be done regularly after treatment (Kgatle et al., 2016; Vorster et al., 2015). The rise in PSA levels after surgery, radiation or during hormonal treatment could be an indication that the cancer is recurring or continuing to grow (Afaq et al., 2018; Bouchelouche et al., 2010; Vorster et al., 2015). If the recurrence of the cancer happens early with a rapid rise in PSA after localised treatment, the more likely that the cancer cells were already distant from the site of the prostate (Afaq et al., 2018; Vorster et al., 2015; UCSF Medical Center).

2.3.3 Digital rectal examination

Digital rectal examination (DRE) is a physical examination of the prostate wherein a doctor inserts a gloved, lubricated finger into the male patient’s rectum to feel for any irregular or abnormally hard areas in the prostate that might be cancer.

2.4 Diagnosis of prostate cancer

PSA and DRE are screening tests which cannot diagnose prostate cancer, but can indicate whether further tests to diagnose or confirm prostate cancer need to be done (Kgatle et al., 2016). There are a few diagnostic tests which can be done (Table 2.2).

Table 2.2: Screening and diagnostic tests which are done to determine the presence of prostate

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* PC: Prostrate Cancer, DRE: digital rectal exam, PSA: prostate specific antigen, TRUS: transrectal ultrasound, CAT: computerized axial tomography, MRI: magnetic resonance imaging, PET: positron emission tomography

2.4.1 Transrectal ultrasound

Transrectal ultrasound (TRUS) is a diagnostic technique which uses sound waves to create an image of the prostate. A probe which gives off sound waves is inserted into the rectum of the patient (Bouchelouche et al., 2010). The probe picks up echoes which are created by the prostate when it receives the sound waves from the probe (Bouchelouche et al., 2010). A computer then creates the pattern of echoes into an image of the prostate, which is viewed on a video screen.

The procedure takes less than 10 min. This test is done when high PSA levels are detected, when the DRE results are abnormal, as a guide during some treatment methods such as brachytherapy (internal radiation therapy) or cryosurgery and it can be used to measure the size of the prostate gland (Bouchelouche et al., 2010). TRUS is also used during a prostate biopsy to guide the needles into the correct area of the prostate (American Cancer Society; Bouchelouche et al., 2010).

2.4.2 Prostate biopsy

A prostate biopsy is usually done when the results of a PSA or DRE test suggest the presence of prostate cancer and confirmation or a diagnosis needs to be made (Kgatle et al., 2016). The procedure involves removing a sample of body tissue and viewing it under a microscope. During a TRUS examination test, a thin, hollow needle is inserted through the wall of the rectum into the prostate gland (Bouchelouche et al., 2010). As the needle is removed, it contains a small cylinder of prostate tissue sample. This process is repeated about 6 to 18 times (Bouchelouche et al., 2010). A biopsy usually takes 10 min and can be done in a doctor’s rooms during consultation.

2.4.3 Bone scan

A bone scan is usually done where there are signs of increased PSA levels (>15 ng/ml) and aggressive cancer (Vorster et al., 2015; UCSF Medical Center). It can also be done in cases of

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bone pain, a large tumour and a high Gleason grade (a prostate cancer grading system) (Vorster

et al., 2015). Bone scans involve injecting radioactive material (radiotracers) into the body

which will subsequently be taken up by diseased bone cells (Bouchelouche et al., 2010). A special camera detects the radioactivity and creates an image of the skeleton. Disease bone scan images may suggest that metastatic cancer is present (Kgatle et al., 2016; Vorster et al., 2015). A bone scan may not detect very small metastases (Bouchelouche et al., 2010).

2.4.4 Computerized axial tomography scan

A computerized axial tomography (CAT) scan can often help determine if prostate cancer has spread to nearby lymph nodes (Kgatle et al., 2016). This procedure allows the use of a rotating x-ray beam to make a series of images of the body used to create a detailed cross-sectional image. These images display abnormally enlarged pelvic lymph nodes and/or the spread of the cancer to other organs (Bouchelouche et al., 2010). Before a scan, a patient drinks or is injected with a contrast agent before the first set of images is taken, to help contrast the intestines from the tumours and other organs in the body. This test is usually done if PSA levels are elevated above 20 ng/ml, including evidence of a large tumour and/or a high Gleason grade (Sathekge

et al., 2018(a)). A CAT scan creates detailed images of soft tissue in the body. CAT scans,

although like x-rays, take longer than regular x-ray acquisition (American Cancer Society).

2.4.5 Magnetic resonance imaging scan

Magnetic resonance imaging (MRI) scans produce clear images of the prostate and can tell whether the cancer has spread to the seminal vesicles or other nearby structures (Bouchelouche

et al., 2010). These scans give information which can be used in planning the treatment of the

patient. MRI scans use the same principle as CAT scans except that they use magnetic fields rather than x-rays to create images of selected areas of the body. MRI is not really effective in diagnosing microscopic or very small cancers. Like CAT scans, MRI does not give useful information on newly diagnosed prostate cancer that may be confined to the prostate (Bouchelouche et al., 2010). MRI scans often take up to an hour which is longer than a CAT scan (UCSF Medical Center).

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2.5 Treatment for prostate cancer

The selection of treatment options for prostate cancer is dependent on a few factors, such as a patient’s age, their general health status, the type of cancer, whether the cancer has spread or not and whether they have a previous history of prostate cancer treatment (Kgatle et al., 2016). Current treatment options include radiation therapy, surgery, and chemotherapy, among others (Afaq et al., 2018). Surgery and radiation treatment is reported to be quite effective in the early stages of prostate cancer (Afaq et al., 2018; Xu et al., 2014). However, and unfortunately, there is no effective treatment for prostate cancer which has metastasised (Emmett et al., 2017; Sathekge et al., 2018(a)). A brief discussion of the three standard treatment options for men with organ-confined prostate cancer follows. Other treatment options which are available will also be discussed later in this chapter.

2.5.1 Active surveillance

Active surveillance, also known as “watchful waiting” is the monitoring of the disease in selected patients (Bouchelouche et al., 2010. Here, the prostate cancer is monitored regularly with PSA tests, clinic evaluation and prostate biopsies to ensure that the cancer does not become aggressive (Kgatle et al., 2016). This is often recommended if the cancer does not cause any symptoms and is expected to grow very slowly. This form of treatment is often selected for men who have other health problems and are older. Older men who have the disease may never require treatment because prostate cancer generally spreads very slowly.

2.5.2 Surgery

Treatment through surgery involves removing the whole prostate, including the seminal vesicles through a procedure known as radical prostatectomy (Emmet et al., 2017; Kgatle et

al., 2016). A surgeon makes skin incisions in the lower abdomen in order to remove the

prostate (Afaq et al., 2018).

2.5.3 Radiation therapy

Radiation treatment uses x-rays or gamma rays to kill cancer cells through either non-invasive or minimally invasive exposure to radiation. Table 2.3 shows the three main types of radiation

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therapies that may be recommended. Each patient receives a treatment plan depending on their symptoms, their overall health and the nature of the cancer. Radiation therapy is usually opted for under the following conditions (Kgatle et al., 2016; Sathekge et al., 2018(a)):

 As treatment for low-grade cancer that is still confined to the prostate gland  If the cancer was not completely removed or has recurred in the prostate area after

surgery

 As part of treatment in conjunction with hormone therapy for cancer that has metastasised into nearby tissue

 If the cancer is advanced

 To reduce the size of the tumour and to give relief from possible future symptoms

External beam radiation and brachytherapy (internal radiation) are the main and most common types of radiation therapies used (Afaq et al., 2018; Xu et al., 2014). External beam radiation therapy allows the use of medication containing radiation to be injected into the body (Kgatle

et al., 2016).

Table 2.3: The three types of radiation therapy (UCSF Medical Center)

*PET: positron emission tomography, SPECT: single photon emission computed tomography

2.6 Radiation theranostics 2.6.1 External beam radiation

External beam radiation also known as external radiation therapy (XRT) is radiation therapy which uses beams of high energy x-rays or electrons which are delivered by a linear accelerator to the region of the patient’s tumour to treat or kill cancerous cells. This technique can be used as a diagnostic and treatment tool to cure early stage prostate cancer or to help relieve

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symptoms of bone pain (if the cancer has already disseminated to bones) (Benešová et al., 2015). To reduce side effects in patients, an accurate dose of the radiation needed is calculated and then the radiation beams are aimed at the outlined target in order to acquire images of the areas which are affected (Velikyan, 2014) by the prostate cancer. In most cases, doctors would rather give higher doses of radiation to the prostate gland while reducing the exposure of radiation to nearby healthy tissue.

2.6.1.1 Positron emission tomography

PET is an imaging technique in nuclear medicine which applies the use of radiotracers (or radiopharmaceuticals), a special camera and a computer to evaluate organ and tissue function (Boschi et al., 2013). PET detects early onset of a disease because it can identify body changes at a cellular level (Sathekge et al., 2018(a); Velikyan, 2014). Nuclear medicine techniques, namely PET and/or positron emission tomography/computed tomography (PET/CT) imaging procedures, are non-invasive and painless (with the exception of intravenous (IV) injections) and are used to diagnose and evaluate medical conditions (Kgatle et al., 2016; Velikyan, 2014). Positron emission is based on the theory that positrons undergo instant annihilation when they collide with an electron (matter-antimatter annihilation). This results in the production of two high-energy gamma rays that exit the scene of the annihilation in exactly opposite directions. For neutron deficient radionuclides, there are two possibilities of decay which are positron emission or electron capture. Positron decay is only possible if an energy of 1022 keV or more is available, otherwise electron capture will occur. In practice a surplus in energy is required before a large amount of the decay occurs by positron decay channel instead of electron capture. The neutrino is a particle with zero rest mass which shares its kinetic energy with the positron or the neutron. The positron is slowed down in the tissue by collisions: at the end of track a hydrogen like atom known as a positronium, is formed by the positron and an electron. The positron and electron are antiparticles and so they will annihilate. In the annihilation process, two gamma-quanta of 511 keV are generated in a back-to-back or co-linear manner. In this way both conservation laws are obeyed. A three-quanta annihilation is also possible within the two conservation laws. The three-quanta annihilation only happens if the formed positronium is in its triplet state, which is rare, with a half-life of 7 μs. In the single state the positronium decays with a lifetime of 8 ns. To image the annihilation radiation one should profit from its unique properties at 511 keV co-linear (180°).

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Depending on the type of test and radiotracer being employed, the radiotracer is either inhaled as a gas, orally ingested or intravenously injected. This radiotracer then accumulates in organs or in the body and emits high energy radiation (Weineisen et al., 2015). The radiation is subsequently detected by an imaging device (or camera) that produces images which provide molecular information (Weineisen et al., 2015). In recent times PET images have been superimposed with computed tomography (CT) (Ebenhan et al., 2017; Sathekge et al., 2018(b)) or magnetic resonance imaging (MRI) through what is known as image fusion, to allow information from two different tests to be correlated and interpreted on a single image (Velikyan, 2014). Fused images supposedly give better information leading to a more accurate diagnosis (Ebenhan et al., 2018). Additionally, bimodal systems such as PET/CT and PET/MRI are more effective due to the combination of the functional image of PET with the morphological image of CT or MRI.

PET scans would thus provide information on organ or body functions (i.e. blood flow, oxygen consumption and glucose metabolism) (Sathekge et al., 2018(a)). This would, for example provide information on the activity of an organ. CT scans produce multiple images of the inside of the body. This technique provides anatomic or morphological information (i.e. the location, size and shape of an organ).

Before a new drug is administered in patients, a lot of data has to be collected from animal studies to obtain information such as dose, route of administration, biodistribution of the drug, point of excretion and toxicity. Animal model-based research therefore pose a demand for a small animal PET for collection of data for medical research (Yao et al., 2012). Small animal PET, well known as microPET, is a technique used for the imaging of small animals such as mice and rats using a small, high resolution PET scanner to identify critical or target organs. Mice and rats can host a number of human diseases, which make them ideal subjects for animal studies (Yao et al., 2012). In recent times, detection of prostate cancer lesions using PET imaging of the prostate specific membrane antigen (PSMA) has been extensively studied with respect to its clinical impact (Eder et al., 2014; Sathekge et al., 2018(a)). PSMA serves as or is a cell surface target which is suitable for imaging metastatic lesions because it is expressed in almost all prostate cancer cells (Afaq et al., 2018; Schäfer et al., 2012). This will be discussed in more detail later in this chapter.

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There are a few common uses for PET and PET/CT procedures (Afaq et al., 2018; Sathekge et

al., 2018(a); UCSF Medical Center), namely to:

 Detect cancer

 Determine whether a cancer has disseminated  Determine if a cancer has recurred

 Determine blood flow to and from the heart  Assess the efficiency of a cancer treatment plan  Map out heart function and the brain

 Evaluate brain abnormalities and other nervous system disorders

2.6.2 Radiotracers used for PET

In cases of metastatic, poorly differentiated and hormone-refractory carcinoma, there is a demand for more effective treatment options (Benešová et al., 2015). Prostate cancer targeting based on low molecular weight radio-ligands could possibly offer much more accurate and rapid visualisation, improved staging and effective radiotherapy (Benešová et al., 2015). Based on small molecules, a few PET radiotracers have been investigated for prostate cancer imaging, namely fluorine-18 (18F) (Vorster et al., 2015), carbon-11 (11C) and peptidyl radiotracers based on the gastrin-releasing peptide receptor and PSMA (Benešová et al., 2015). Gallium (III) (Ga(III)) complexes are becoming a favourable alternative to iron (III) (Fe(III)) complexes as PET based anticancer agents because of their similarities (Banerjee et al., 2010). Positron-emitting versions of Ga(III) can be employed in tumour imaging for cancer diagnostic purposes (Sathekge et al., 2018(a)). Gallium-68 (68Ga)-labelled peptides have attracted interest in cancer imaging as a result of the physical characteristics of the isotope (Table 2.4) (Banerjee et al., 2010; Sathekge et al., 2018(a); Velikyan, 2014).

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Table 2.4: Some commonly used radionuclides used in radiotherapy, PET and SPECT,

including their decay properties and mode of production (Velikyan, 2014)

Radionuclide Half-life Emax (keV) Radiation Production

Positron emitters 11_C _{20.3 min} ₉₆₁ _β+_{(100 %)} _Cyclotron 64_Cu _{12.8 h} ₆₅₆ _β+_{(19 %)} _Cyclotron 18_F _{110 min} ₆₃₄ _β+_{(97 %)} _Cyclotron 66_Ga _{9.5 h} ₄₁₅₃ _β+_{(56 %)} _Cyclotron 68_Ga _{67.6 min} _{1899, 770} _β+_{(89 %)} _Generator 124_I _{4.17 d} ₂₁₀₀ _β+_{(23 %)} _Cyclotron Gamma emitters 67_Ga _{78.26 h} _{91, 93, 185, 296, 388} _γ _Cyclotron 111_In _{67.9 h} _{245, 172} _γ _Cyclotron 99m_Tc _{6.0 h} ₁₄₁ _γ _Generator Therapeutic radionuclides

125_I _{60 d} ₃₅₀ _{Auger electrons} _Reactor

131_I _{8.0 d} ₁₈₁₀ _β- _Fission

177_Lu _{6.71 d} ₅₀₀ _β- _Reactor

90_Y _{64.0 h} ₂₂₇₀ _β- _Generator

2.6.2.1 Gallium-68

68_{Ga is easily available from a portable in-house}68_Ge/68_{Ga generator (Ebenhan et al., 2017).} 68_{Ga is a convenient alternative to cyclotron-based isotopes such as}18_{F and}124_{I, especially}

because it is readily available (Umbricht et al., 2017). Germanium-68 (68_{Ge) has a half-life of}

270.8 days (Banerjee et al., 2010; Velikyan, 2014). 68_{Ga emits positron decay (89 % of its}

total decay by β+_{emission) (Banerjee et al., 2010; Müller, 2013). The maximum positron}

energy of 68Ga is 1.92 MeV (mean = 0.89 MeV), which is higher than that of 18F (Eβmax =

0.63 MeV; mean = 0.25 MeV) (Banerjee et al., 2010). The low energy of 68Ga positron emission (Eβmax = 635 keV; 2.2 mm mean range in matter) serves as an advantage for better

resolution of PET images and quantification of biochemical processes in vivo (Malik et al., 2015). 68Ga has a half-life of 68 min (Malik et al., 2015). There is, however drawbacks to

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using 68Ga eluate for the radiolabelling of peptides which include breakthrough of the long-lived parent radionuclide 68Ge, the high HCl concentration (0.1-1 M used for 68Ga elution) and the high eluate volume (Boschi et al., 2013). Subsequently, there are metallic impurities such as Fe3+ (from the column material) and Zn2+ (by-product of the decay of 68Ga, Ti4+) which could be present in the eluate (Boschi et al., 2013) and ultimately result in lowered specific activity and yield of 68Ga. Impurities affect the yield of 68Ga and the specific activity of the labelled product. One of the ways to reduce impurities would be to fractionate the eluate since about two-thirds of the total 68_{Ga activity elutes within a 1-2 ml activity peak (Boschi et al.,}

2013; Mokaleng et al., 2015). The Ga(III) ion forms a stable complex (formation constant logKML = 21.33) with DOTA, which is commercially available (Mokaleng et al., 2015).

2.7 Alternative treatment

Table 2.5 shows a summary of other treatment options which are used for prostate cancer therapy. Like most treatment methods, these aim to kill or slow down the growth of prostate cancer cells and may probably offer benefits with less side effects. Some of these treatments are only available through clinical trials that are designed to test their effectiveness as compared to currently available treatment options.

Table 2.5: Alternative prostate cancer treatment options (UCSF Medical Center)

*ADT: androgen deprivation therapy

2.8 Prostate specific membrane antigen

Prostate specific membrane antigen (PSMA) is a type of protein (Table 2.6) which is expressed in all forms of prostate cells and carcinoma (Sathekge et al., 2018(a)). It is a transmembrane glycoprotein glutamate carboxypeptidase II (GCPII) (Malik et al., 2015; Wiehr et al., 2014),

Evaluation of the effect of colloidal systems on biodistribution of selected prostate cancer radiopharmaceuticals